智利江蓠中硝酸还原酶的体外测定及光调节

In vitro assay and light regulation of nitrate reductase in red alga Gracilaria chilensis.

作者信息

Chow Fungyi, de Oliveira Mariana C, Pedersén Marianne

机构信息

Instituto de Biociências, Universidade de São Paulo, CP, CEP, São Paulo, SP, Brazil.

出版信息

J Plant Physiol. 2004 Jul;161(7):769-76. doi: 10.1016/j.jplph.2004.01.002.

DOI:10.1016/j.jplph.2004.01.002

PMID:15310065

Abstract

Nitrate reductase (NR) is the first enzyme in the nitrogen assimilation pathway. The in vitro NR activity of Gracilaria chilensis was assayed under different conditions to reveal its stability and biochemical characteristics, and an optimized in vitro assay is described. Maximal NR activities were observed at pH 8.0 and 15 degrees C. The apparent Km value for NADH was 8 microM and for nitrate 680 microM. Crude extracts of G. chilensis stored at 4 degrees C showed a 50% decrease of NR activity after 24 h. The highest NR activity value (253.20+/-2.60 x 10(-3) U g(-1)) was obtained when 100% von Stosch medium (500 microM NO3-) was added before extraction of apical parts. Algae under light:dark cycles of 12:12h exhibited circadian fluctuation of NR activity and photosynthesis with more than 2 times higher levels in the light phase. No evidence of endogenous diel rhythm controlling NR activity or photosynthesis was observed. Light pulses lasting 10 or 60 min during the darkness increased the NR activity by 30% and 45%, respectively. The results indicate that NR and photosynthesis are regulated mainly by light and not by a biological clock.

摘要

硝酸还原酶（NR）是氮同化途径中的第一种酶。对智利江蓠的体外NR活性在不同条件下进行了测定，以揭示其稳定性和生化特性，并描述了一种优化的体外测定方法。在pH 8.0和15℃时观察到最大NR活性。NADH的表观Km值为8 microM，硝酸盐的表观Km值为680 microM。在4℃下储存的智利江蓠粗提取物在24小时后NR活性下降了50%。在提取顶端部分之前添加100%的冯·施托希培养基（500 microM NO3-）时，获得了最高的NR活性值（253.20±2.60×10-3 U g-1）。处于12:12小时光暗循环下的藻类表现出NR活性和光合作用的昼夜波动，光期水平比暗期高2倍以上。未观察到内源性昼夜节律控制NR活性或光合作用的证据。在黑暗期间持续10或60分钟的光脉冲分别使NR活性提高了30%和45%。结果表明，NR和光合作用主要受光调节，而非生物钟调节。